Sieve valves, microfluidic circuits, microfluidic devices, kits, and methods for isolating an analyte

Abstract

The invention further provides methods of using the device for the analysis of samples comprising cells.

What is claimed is: 
     
       1. A method of isolating an analyte, the method comprising:
 (i) loading a sample into a holding chamber of a microfluidic circuit, wherein the microfluidic circuit comprises:
 one or more holding chambers in fluid communication with an input valve; 
 a mixing circuit comprising a plurality of chambers in fluid communication with the one or more holding chambers, wherein the one or more holding chambers and/or the mixing circuit comprises a capture substrate for capturing and isolating the analyte or component; 
 one or more sieve valves positioned between the holding chamber and the mixing circuit and positioned in at least partial fluid communication with one or more of the plurality of chambers of the mixing circuit, and 
 one or more sieve valves positioned in at least partial fluid communication with one or more of the plurality of the chambers of the mixing circuit, 
 wherein each of the one or more sieve valves includes: 
 a substrate defining a channel; 
 a flexible membrane adapted and configured for deployment at an intersection with the channel; wherein one or more protrusions extends into the channel from the substrate and is situated opposite the flexible membrane, the one or more protrusions defining a plurality of recesses extending beyond the intersection between the channel and the flexible membrane; and 
 an output valve; 
 
 (ii) passing the sample into the mixing circuit; 
 (iii) capturing the analyte on the capture substrate in the mixing circuit; 
 (iv) washing the capture substrate to remove uncaptured components; and 
 (v) releasing the analyte from the capture substrate. 
 
     
     
       2. The method of  claim 1 , wherein the capture substrate is loaded into the microfluidic circuit, the holding chamber, and/or the mixing circuit. 
     
     
       3. The method of  claim 1 , wherein the capture substrate comprises a bead, microbead, surface of the microfluidic circuit, or a capture reagent. 
     
     
       4. The method of  claim 1 , wherein the microfluidic circuit is contained in a microfluidic device. 
     
     
       5. The method of  claim 4 , wherein the microfluidic device comprises a plurality of microfluidic circuits which lie in a plurality of laminar layers of the microfluidic device. 
     
     
       6. The method of  claim 5 , wherein the plurality of microfluidic circuits of the microfluidic device span across a plurality of laminar layers of the microfluidic device. 
     
     
       7. The method of  claim 1 , further comprising opening at least one of the sieve valves to remove the capture substrate from the microfluidic circuit, the holding chamber, and/or the mixing circuit. 
     
     
       8. The method of  claim 1 , wherein the method further comprises:
 activating the sieve valve positioned between the holding chamber and the mixing circuit in order to retain the capture substrate and the sample within the holding chamber while permitting fluid to pass through the sieve valve. 
 
     
     
       9. The method of  claim 3 , wherein the capture reagent comprises an antibody, carboxylic acid, cation, anion, cationic group, anionic group, hydrophobic group, magnetic material, protein, ligand, nucleic acid, affinity agent, or a combination thereof. 
     
     
       10. The method of  claim 1 , wherein the sample comprises one or more of a single cell, a plurality of cells, a tissue, an organelle, a particle, an organism, a virus, a microorganism, a spore, a fungus, a nucleic acid molecule, a polypeptide, a carbohydrate, a lipid, or a small molecule. 
     
     
       11. The method of  claim 10 , wherein the sample comprises a prokaryotic cell, a eukaryotic cell, or a plurality thereof. 
     
     
       12. The method of  claim 1 , wherein the analyte comprises one or more of a nucleic acid molecule, polypeptide, carbohydrate, lipid, or small molecule. 
     
     
       13. The method of  claim 1 , further comprising:
 loading one or more reagents for modifying the analyte in the microfluidic circuit; 
 mixing the one or more reagents and the analyte in the mixing circuit to modify the analyte; and 
 removing the modified analyte from the microfluidic circuit. 
 
     
     
       14. The method of  claim 13 , wherein the modifying comprises fixing a label, reporter, nucleic acid molecule, polypeptide, or synthetic molecule to the analyte. 
     
     
       15. The method of  claim 13 , wherein the analyte is a nucleic acid molecule and the method comprises:
 loading one or more reagents for fragmenting the nucleic acid molecule in the microfluidic circuit; 
 mixing the one or more reagents and the nucleic acid molecule in the mixing circuit to fragment the nucleic acid molecule; 
 removing the nucleic acid fragments from the microfluidic circuit; and optionally, 
 fixing an amplification adaptor to the 5′ and 3′ ends of the nucleic acid fragments. 
 
     
     
       16. The method of  claim 15 , further comprising:
 contacting the nucleic acid fragments with primers that hybridize to the amplification adaptors inside the mixing circuit; and 
 amplifying the nucleic acid fragments inside the mixing circuit. 
 
     
     
       17. The method of  claim 16 , wherein the amplifying comprises polymerase chain reaction (PCR) amplification, helicase-dependent amplification (HAD), nicking enzyme amplification reaction (NEAR), loop mediated isothermal amplification (LAMP), rolling circle amplification (RCA), or recombinase polymerase amplification (RPA). 
     
     
       18. The method of  claim 17 , wherein the nucleic acid fragments provide a sequence library pool of individual samples having a coefficient of variation (COV) of 0.5 or less in their respective quantities. 
     
     
       19. A method of isolating an analyte, the method comprising:
 (i) loading a sample into a holding chamber of a microfluidic circuit, wherein the microfluidic circuit comprises: 
 one or more holding chambers; 
 a mixing circuit comprising a plurality of chambers in fluid communication with the one or more holding chambers, wherein the one or more holding chambers and/or the mixing circuit comprises a capture substrate for capturing and isolating the analyte or component; 
 one or more sieve valves positioned between the holding chamber and the mixing circuit and positioned in at least partial fluid communication with one or more of the plurality of chambers of the mixing circuit; 
 one or more sieve valves positioned in at least partial fluid communication with one or more of the plurality of the chambers of the mixing circuit; 
 wherein each of the one or more sieve valves includes: 
 a substrate defining a channel; 
 a flexible membrane adapted and configured for deployment at an intersection with the channel; wherein 
 one or more protrusions extends into the channel from the substrate and is situated opposite the flexible membrane, the one or more protrusions defining a plurality of recesses extending beyond the intersection between the channel and the flexible membrane; and 
 an output valve; 
 (ii) passing the sample into the mixing circuit; 
 (iii) capturing the analyte on the capture substrate in the mixing circuit; 
 (iv) washing the capture substrate to remove uncaptured components; and 
 (v) releasing the analyte from the capture substrate.